The detection and/or quantitation of specific nucleic acid sequences is an important technique for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. Such procedures are also useful in detecting and quantitating microorganisms in foodstuffs, water, industrial and environmental samples, seed stocks, and other types of material where the presence of specific microorganisms may need to be monitored. Other applications are found in the forensic sciences, anthropology, archaeology, and biology where measurement of the relatedness of nucleic acid sequences has been used to identify criminal suspects, resolve paternity disputes, construct genealogical and phylogenetic trees, and aid in classifying a variety of life forms.
The ability to amplify ribonucleic acid (RNA) is an important aspect of efforts to elucidate biological processes. Amplification of the total cellular mRNAs prepared from any cell or tissue is important for gene expression profiling. Total cellular mRNA represents gene expression activity at a defined time. Gene expression is affected by cell cycle progression, developmental regulation, response to internal and external stimuli and the like. The profile of expressed genes for any cell type in an organism reflects normal or disease states, response to various stimuli, developmental stages, cell differentiation, and the like. Non-coding RNAs have been shown to be of great importance in regulation of various cellular functions and in certain disease pathologies. Such RNAs are often present in very low levels. Although analysis of non-amplified mRNA is feasible, a significant amount of starting mRNA can be required. Thus, amplification methods capable of amplifying low abundance RNAs, are of great importance.
RNA amplification is commonly performed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method and variations thereof. These methods are based on replication of RNA by reverse transcriptase to form single stranded DNA complementary to the RNA (cDNA), which is followed by amplification techniques such as polymerase chain reaction (PCR) or linear isothermal amplification to produce multiple copies of single or double stranded DNA, or RNA. However, the total amount of sample RNA that is available is frequently limited by the amount of biological sample from which it is derived. Biological samples are often limited in amount and precious. Moreover, the amount of the various RNA species is not equal; some species are more abundant than others are, and these are more likely and easier, to analyze. The ability to amplify RNA sequences enables the analysis of less abundant, rare RNA species. The ability to analyze small samples, by means of nucleic acid amplification, is also advantageous for design parameters of large scale screening of effector molecule libraries, for which reduction in sample volume is a major concern both for the ability to perform very large scale screening or ultra high throughput screening, and in view of the limiting amounts of library components. Methods of amplification from RNA templates have been described, for example in U.S. Pat. No. 6,946,251.
RNA in biological samples is often in the presence of DNA. Amplification of the target RNA in the presence of DNA results in unwanted amplification products as described herein. It is desirable to prevent these unwanted products that originate from initiation on DNA because they may interfere with analysis of target RNA amplification products, result in erroneous conclusions and affect the amplification yield from the target RNA. Purification of RNA from DNA results in reduced yield and/or RNA quality. Therefore, it is highly desirable to develop improved amplification methods of target RNA in the presence of DNA. Moreover, the ability to selectively amplify RNA in a sample comprising total nucleic acid from a biological sample will also assist in the development of procedures and methods for selective amplification in situ as well as directly from stabilized cell lysates. This is especially useful when there are minute amounts of sample for analysis.
Therefore, there is a need for improved RNA amplification methods that overcome drawbacks in existing methods. The invention provided herein fulfills this need and provides additional benefits.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.